Blohm, Gabriela Maxine

THE EFFECTS OF POPULATION STRUCTURE AND RESOURCE LEVELS

ON THE PREVALENCE OF A VERTICALLY TRANSMITTED VIRUS

THEORETICAL BACKGROUND

The prevalence of a pathogen depends on intrinsic factors such as its mode of

transmission and host immunity; and extrinsic factors such as the spatial
arrangement of hosts and the availability of growth-limiting resources. To date, the
majority of studies on the evolution and control of infectious diseases have invoked
mainly intrinsic factors to predict the prevalence of a pathogen (e.g. SIR models
and Individual-based models) (Messinger 2009). For example, the tradeoff theory
postulates that the extinction rate of a pathogen is based on 2 key components:
virulence (defined as the deleterious effect of a pathogen on the fitness of its host)
and rate of transmission. Its primary assumption is that a pathogen’s probability of
infecting other hosts decreases when virulence is high. The tradeoff theory has
provided a useful mechanistic foundation for generating testable predictions about
the evolution of virulence. A limitation of this theory, however, is that it fails to
explain the observed ubiquity of high-virulence pathogens; high virulence
commonly leads to extinction in simulation studies, yet it is a common strategy
among infectious diseases worldwide (Messinger 2009).

Conceptually, it is useful to imagine that the intrinsic components of a host-

pathogen system operate within a set of rules that are set by extrinsic factors. The
evolutionary outcome of a pathogen invasion may vary depending on the
environmental conditions of the system (e.g. resource availability). The body of
empirical evidence to support this idea is growing. For example, the spatial
arrangement of hosts can give rise to unequal probabilities of pathogen
transmission, producing a network of ‘hotspots’ (Thompson 1994) where the rate of
coevolution between the host and pathogen varies in space.

When choosing the spatial scale over which a given set of parameter values are
recorded in the environment, it is important to understand 1) the distance over
which the variance of a given parameter is autocorrelated in space; and 2) the
distance over which a key driver of fixation (e.g. selection vs. drift) might shift.
Knowing the effect of space on our ability to obtain values that are representative of
both the mean and variance of a parameter is a sound first step towards polishing
the virulence-transmission tradeoff curve that is calculated for a particular host-
pathogen system. The first aim of this study is to quantify the local and regional
scale variation in the prevalence of a pathogen (details of the study system and
data collection methods are explained in subsequent sections).

An additional extrinsic component of host-pathogen dynamics is the availability

of growth-limiting resources. Several recent studies in plant communities have
found that pulses of nitrogen and phosphorous can alter the short-term outcome of
a pathogen invasion (Blumenthal et al 2009). One might predict that a pulse of

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nutrients confers an immunological advantage to the host; however the reverse can
be the case depending on the stoichiometric ratio of the nutrient pulse (extrinsic)
and the host’s immunological response to nutrient inputs (intrinsic). Our current
agricultural practices have produced large-scale shifts in the distribution of key
nutrients such as nitrogen and phosphorous. For example, the rate of nitrogen
fixation has doubled since the development of the Haber-Bosch method.
Particularly, aquatic and coastal ecosystems are exhibiting high levels of
phosphorous due to agricultural runoff (Harvell et al 2002). Advancing our
understanding of host-pathogen coevolution will likely require that we qualify our
predictions with the knowledge of N and P effects on virulence. The current
mismatch between the predictions of the tradeoff theory and the observation that
high-virulence pathogens are common may be resolved by adding a resource-level
component to our models.

The empirical evidence that describes the impact of space and resource levels
on pathogen prevalence is still relatively patchy. It is therefore challenging to 1)
identify and 2) incorporate new important drivers of pathogen virulence without
compromising the simplicity of our models. Studies that combine experiments,
observational surveys and simulations provide us with the advantage of
approaching a question from several perspectives and allow us to identify the key
components of an interaction. I outline a study that employs this approach with the
goal of understanding how space and resource levels (extrinsic mechanisms) can
affect the evolution of virulence.

STUDY SYSTEM

Vertical transmission is common among vectors of human pathogens (e.g. West

Nile Virus, Ross River, Dengue fever) and involves the transmission of a pathogen
from parents to offspring. Understanding the ecology and evolution of this mode of
transmission will inform strategies for controlling several diseases that constitute
heavy economic burdens in many countries. Additionally, these studies will
contribute to our understanding of the evolution of the virulence-transmission
tradeoff, given that this mode of transmission exhibits several constraints that differ
than those faced by horizontally transmitted pathogens. For example, virulence is
more likely tied to ontogeny in vertically transmitted pathogens than in horizontally
transmitted pathogens.

Sigma virus is a vertically transmitted rhabdovirus that infects Drosophila

melanogaster worldwide (Carpenter 2007). It is biparentally transmitted and results
in reduced overwintering survival, reduced female fecundity, and high sensitivity to
CO2 (infected flies do not recover from CO2 exposure in the laboratory). An allele
associated with sigma virus resistance has also been identified (Wayne et al 1996).
Wild populations of D. melanogaster exhibit infection rates that vary between 0%
and 14.9% (Table 1); during the summer of 2009, however, we found that 30% of
the population of D. melanogaster in SE Georgia is infected with sigma virus

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(Figure 1), representing nearly twice the highest viral prevalence obtained from
the studies in 2005.

Table 1. From Carpenter et al 2007.

Figure 1. Proportion of D. melanogaster infected with sigma virus along a 52-mile transect between Eatonton and
Athens, GA during June-September 2009. Error bars are approximate 95% confidence intervals (calculated as
2*Standard Error of the mean). The average infection rate ranged from 15% to 38%. Both of these values were
collected in the central region of the transect. Sample size is indicated by the numbers at the top of each standard
error bar.

To understand these sharp contrasts in viral prevalence, we first calculated

several intrinsic parameters in the lab. Through laboratory CO2 assays, we
quantified the rate of sigma virus transmission: infected females transmit the
disease to 100% of their offspring while infected males transmit the virus to 40% of
their offspring (Figure 2). We also found that infection with sigma virus reduces the
fecundity of females by approximately 31%.

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Rate of sigma virus transmission by females and males along the

Eatonton-Athens transect

Figure 2. The transmission rate of sigma virus by males and females for each sampled location along the
Eatonton-Athens transect. We found a significant difference between male and female rate of transmission,
measured as the proportion of offspring that were infected. The data were obtained from crosses between wild
infected individuals and virgin laboratory uninfected individuals.

We then constructed a discrete-time model that utilizes the parameters obtained

in the laboratory to predict the prevalence of sigma virus in the field:

Pt+1 = (1-Pt)(nieF + nueM) + Pt2 bni

where P represents the proportion of individuals that are infected at time t; n represents the number of D.
melanogaster hosts that are either i infected or u uninfected; e represents the transmission rate by either F
females or M males and b represents the proportion of individuals that are progeny of both infected parents.

We found that the model fixes at an infection frequency above 60%, which is
higher than what is observed in nature. Several possible explanations exist for this
discrepancy. We aim to investigate the following hypotheses:

Distance Hypothesis: 1) Viral prevalence and 2) the shape of the

virulence-transmission tradeoff curve for sigma virus can be explained by
the spatial correspondence between D. melanogaster and sigma virus,
assuming there is isolation by distance among populations.

Phosphorous Hypothesis: 1) Viral prevalence and 2) the shape of the

virulence-transmission tradeoff curve for sigma virus can be explained the
C:N:P ratio of the food resource for D.melanogaster.

PROJECT DESCRIPTION

To test the Distance hypothesis, we will conduct a series of intensive field

surveys throughout the Eastern United States in the following locations, in which the
presence of sigma virus has been detected:

Eatonton, GA: Continue surveys along the transect to estimate local-scale

patterns in sigma virus prevalence. We will return the flies to the lab and store them
at -80C for subsequent quantification of viral titer using QPCR techniques that have
been developed in the laboratory by Dr. Marta Wayne and optimized by Jena

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Chojnowski. Additionally, we will sequence 82 SNPs (loci and methods in Dieringer

et al 2008) in D. melanogaster, as well as hyper-variable regions in the sigma virus
genome for further association mapping using STRUCTURE. We will develop a
mechanistic metric of correspondence between D. melanogaster and sigma virus
assuming isolation by distance and determining whether sex-biased dispersal exists
in these populations.

We will set traps and store individuals as described above in the following locations:

Apshawa, FL; Wildwood, FL. (Carpenter 2007); Ithaca, NY (Yampolsky 1999);

Raleigh, NC (Bangham 2007); Allentown, Pennsylvania (Bangham 2007); and Aiken,
SC.

Sequence data from these locations will be used to determine large-scale

patterns in genetic variation and test the association parameters obtained from the
intensive sampling in the Eatonton-Athens transect.

To test the Phosphorous hypothesis, we will conduct three types of

crosses in the lab as follows: Infected males + uninfected females; Infected females
+ uninfected males; Infected females + infected males. Each set of crosses will be
exposed to 2 levels of phosphorous: Low and high (see methods in Pletcher et al
2002). We will then calculate the viral titer of all individuals using QPCR to quantify
the effects of nutrient inputs on viral load.

BROADER IMPACTS

The data that have been collected to date were the result of a collaboration
among female undergraduates and graduate students from underrepresented
groups at the University of Florida. The results of our surveys and experiments have
been presented in two conferences. Additionally, the community of peach stand
owners along Highway 441 near Eatonton, GA was an essential component of the
project. We will continue collaborating with the South Georgia community and hope
to expand and diversify our network of collaborators.

BUDGET JUSTIFICATION

1. Travel and lodging during Summer of 2010 $1,000

2. QPCR supplies and reagents $2,000
3. Fly rearing supplies $1,000
4. Sequence analysis supplies and reagents $3,000

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TOTAL $6,000

WORKS CITED

Blumenthal, D., C.E. Mitchell, P. Pysek, and J. Jarosik. 2009. Synergy between
pathogen release and resource availability in plant invasion. Proceedings of the
National Academy of Sciences 106: 19

Carpenter, J.A., D.J. Obbard, X. Maside, and F. Jiggins. 2007. The recent spread of a
vertically transmitted virus through populations of D. melanogaster. Molecular
Ecology 16: 3947-3954.

Dieringer, D., V. Nolte and C. Schlotterer. 2005. Population structure in African

Drosophila melanogaster
revealed by microsatellite analysis. Molecular Ecology 14: 563-573

Messinger, S.M. and A. Ostling 2009. The consequences of Spatial Structure for the
Evolution of Pathogen Transmission Rate and Virulence. The American Naturalist
174: 441-454.

Pletcher et al 2002. Genome-wide transcript profiles in aging and calorically

restricted Drosophila melanogaster. Current Biology 12: 712-723

Thompson, J.N. 1994. The coevolutionary process, University of Chicago Press.

Wayne, M.L., D. Contamine and M. Kreitman. Molecular population genetics of

ref(2)P, a locus which confers viral resistance in Drosophila. Molecular Biology and
Evolution 13: 191-199.